The research proposed is designed to determine the intramitochondrial localization of cytochrome P-450 and the various steroid hydroxylation reactions occurring in adrenal cortex. The aim is to separate, purify and characterize the components required in these cytochrome P-450 catalyzed reactions. Efforts will be mainly directed toward purifying cytochrome P-450, the TPNH-specific flavoprotein dehydrogenase and other - as yet unidentified - components (since rather detailed knowledge of the chemistry of adrenodoxin is already available). The components required for steroid 11 beta-hydroxylation will be compared with those found to be needed for the side chain cleavage of cholesterol. Since the rate of corticosteroid synthesis is determined principally by the cholesterol desmolase reaction, these studies should indicate the unique features of this reaction which allow specific regulation by ACTH.